Quantum dots are nanometer-size semiconductor particles, which emit light when they are excited with energy such as light, and the color of the emitted light depends on the size of the particles. That is, when the size of the particles is decreased to reduce the dimension of the particles, the electronic state density and energy thereof will be varied, and thus the characteristics of the particles will vary depending on the dimension thereof. For example, quantum hall effect, which does not appear in three-dimensional systems, is observed in two-dimensional systems. As used herein, the term “reducing dimension” in the strict sense means that electrons are confined to an area smaller than the de Broglie wavelength. A zero-dimensional quantum dot is not a dot having no area, but rather refers to a sample having a three-dimensional size smaller than the de Broglie wavelength. In quantum mechanics, a wave in all material particles with momentum, that is, the de Broglie wavelength, varies depending on material and is about 10 nm for semiconductor material.
As shown in FIG. 1, a quantum dot is generally in the form of a sphere consisting of a core and a shell and contains, in addition to Zn, S and Cd illustrated in FIG. 1, other various heavy metals.
Methods for preparing quantum dots can be broadly divided into two categories: lithographic methods using a light source such as a laser; and chemical synthesis methods. The synthesis of quantum dots by chemical method has an advantage in that it can produce quantum dots using a relatively simple system compared to the lithographic method, but this method still has a lot of technical problems to be solved in order to produce a large amount of quantum dots in a cost-effective manner. Also, in comparison with quantum dots prepared using the prior bulk method, quantum dots prepared using molecular chemical technique have excellent optical stability, and their optical properties can be controlled so as to emit light at various wavelengths depending on the size, shape and component of nanomaterials. For this reason, it has become possible to apply quantum dot-based nanomaterials in the biological field. This indicates that the quantum dot-based nanomaterials can be widely used as not only biosensors, but also contrast agents for in vivo optical imaging, and the possibility thereof has recently been proven through various studies (Jovin, Nat. Biotechnol., 21:32, 2003; Wu et al., Nat. Biotechnol., 21:41, 2003; Alivisatos, Nat. Biotechnol., 22:47, 2004; Gao et al., Nat. Biotechnol., 22:969, 2004; Lidke et al., Nat. Biotechnol., 22:198, 2004).
As typical examples thereof, techniques capable of tracking the signal transduction of live cells in vitro were developed, and one example thereof is an approach to the HER2/neu pathway. This overcomes the shortcoming of existing organic fluorophores, in that they readily lose their optical stability, such that they cannot track continuous cell changes (Wu et al., Nat. Biotechnol., 21:41, 2003; Lidke et al., Nat. Biotechnol., 22:198, 2004). Also, in order to track erbB/HER receptor-mediated signal transduction in live cells, an epidermal growth factor (EGF) was conjugated to quantum dots and activated through binding to an epidermal growth factor receptor (EGFR). Then, the process of incorporation of the quantum dot conjugate-conjugated EGFR into cells was tracked (Lidke et al., Nat. Biotechnol., 22:198, 2004). This method enabled biological processes in live cells to be observed in real time.
Furthermore, to overcome a shortcoming of insufficient in vivo permeability, semiconductor nanocrystals, which can use wavelengths in the near infrared range, were developed, thus making in vivo image acquisition possible (Gao et al., Nat. Biotechnol., 22:969, 2004). In this method, in order to effectively make quantum dots soluble and, at the same time, effectively deliver quantum dots in vivo, quantum dots (ZnS-capped CdSe) were coated with a triblock copolymer, and a monoclonal antibody is conjugated to the reactive group of the coated polymer.
In addition, to overcome a shortcoming of low in vivo permeability of optical imaging, quantum dots, which can be adjusted to the near infrared wavelength, were developed. For example, quantum dots having the near infrared wavelength were used to optically image the sentinel lymph node in the armpit of mice (Kim et al., Nat. Biotechnol., 22:93, 2004). Such nanomaterials show physical properties different from those of the bulk method as shown in quantum dots, and thus have the potentiality to be used in magnetic resonance imaging.
Meanwhile, methods for treating metal ions in the environment generally include chemical, physical and biological treatment methods. The biological treatment method employs the biological mechanisms of microorganisms themselves, and microbial mechanisms, including biosorption & bioaccumulation, oxidation & reduction, metal-organic complexation and insoluble complex formation, provide an important technical foundation for restoring the environment contaminated with heavy metal (Valls and de Lorenzo, FEMS Microbiol. Rev., 26:327, 2002). Furthermore, with the development of molecular biology, a recent attempt to develop strains having an increased ability to bind heavy metals suggests the possibility to improve prior biological treatment methods. Microorganisms synthesize heavy metal-binding proteins to remove heavy metals in vivo and ex vivo through bioaccumulation, and these proteins are involved in the storage or regulation of concentration of intracellular metal ions.
Recently, it was found that peptides, called phytochelatins, which naturally bind to harmful elements, such as lead, mercury and cadmium, to detoxify these elements, are produced by fungi and plants, exposed to heavy metals. Phytochelatins have a structure of (gamma-Glu-Cys)n-Gly (n=2-7) and accumulate metal ions through the formation of peptide-metal conjugates (Cobbett, Curr. Opin. Plant Biol., 3:211, 2000). In addition, as other kinds of heavy metal-binding proteins, low-molecular proteins called metallothioneins have been much studied, and these proteins have a high content of cysteine and bind to cadmium, zinc, nickel, copper and the like (Hamer, Annu. Rev. Biochem., 55:913, 1986; Wu and Lin, Biosens. Bioelectron., 20:864, 2004).
Meanwhile, patent documents relating to the preparation of quantum dots include: Korean Patent Registration No. 10-0540801, entitled “Method of preparing quantum dots using metal powder”; Korean Patent Registration No. 10-0526828, entitled “Method of preparing quantum dots having uniform distribution by irradiating magnetic membrane or semiconductor membrane with laser”; Korean Patent Registration No. 10-0541516, entitled “Method for forming quantum dots of semiconductor material”; and Korean Patent Registration No. 10-0279739, entitled “Method for forming nanometer-size silicon quantum dots. However, these methods are methods of preparing quantum dots through the physical binding of metal materials.
Having paid attention to that the component of a quantum dot is synthesized by a regular arrangement of cadmium (Cd), selenium (Se), zinc (Zn) and tellurium (Te), the present inventors expressed a heavy metal-binding protein using a bioengineering method. As a result, the present inventors have found that it is possible to synthesize highly economical quantum dots in vivo, and also used the above biological system to prepare quantum dots having optical stability, thereby completing the present invention.